Enzyme Immobilization

Fig. 9. (A) Effect of pH on the stability of invertase Sepabeads EC-EP3-PEI immobilized preparations. (♦) pH 5.0; (•) pH 5.5; (■) pH 7.0; (A) pH 8.5. Enzyme was immobilized at pH 7.0 as described in Subheading 3., with a protein loading of 25 mg protein/g of support. Inactivation of enzyme preparations was carried out at 55°C. (B) Thermal stability of invertase Sepabeads EC-EP3-PEI preparations immobilized at different pH values: enzyme load was 25 mg of protein/g of support. (A) Immobilized enzyme at pH 8.5; (■) enzyme immobilized at pH 7.0; (•) enzyme immobilized at pH 5.5. Inactivation of immobilized enzyme was performed at pH 4.5 and 55°C.

3.9. Stability in the Presence of Cosolvents of Immobilized Enzymes on Polymeric Supports

In the presence of organic media, all industrial enzymes immobilized on sul-fate-dextran supports exhibited certain stabilization. These results show that the use of ionic polymers such as sulfate-dextran may generate a hydrophilic micro-enviroment around the enzymes, preventing their inactivation by partition of the organic solvent molecules. Figure 10 shows that trypsin and chymotrypsin immobilized on the sulfate-dextran support (see Subheading 3.3.) were also much more stable against the action of organic solvents compared to the CMC immobilized preparation. The stability studies were carried out as described in Subheading 3.6.

Bovine Trypsin Thermal Stability

Time (hours) Time (houre)

Fig. 10. Inactivation of different chymotrypsin and trypsin from bovine pancreas sul-fate-dextran derivatives in the presence of cosolvents. Immobilization was performed at pH 7.0 on optimized sulfate-dextran support as described in Subheading 3. Inactivation of derivatives was performed at pH 7.0 at 25°C in the presence of different percentage of dioxane. Other specifications are described in Subheading 3. (A). Stability of trypsin derivatives in presence 30% dioxane. (♦) Optimized sulfate-dextran support immobilized derivative at pH 7.0. (■) Optimized sulfate-dextran support immobilized at pH 5.0. (A) Carboxymethyl support immobilized derivative at pH 7.0. (•) Carboxymethyl support immobilized derivative at pH 5.0. (B) Stability of chymotrypsin derivatives in presence 50% dioxane. (♦) Optimized sulfate-dextran support immobilized derivative pH 7.0. (A) Optimized sulfate-dextran support immobilized derivative pH 5.0. (■) Carboxymethyl support immobilized derivative at pH 7.0. (•) Carboxymethyl support immobilized derivative at pH 5.0.

Time (hours) Time (houre)

Fig. 10. Inactivation of different chymotrypsin and trypsin from bovine pancreas sul-fate-dextran derivatives in the presence of cosolvents. Immobilization was performed at pH 7.0 on optimized sulfate-dextran support as described in Subheading 3. Inactivation of derivatives was performed at pH 7.0 at 25°C in the presence of different percentage of dioxane. Other specifications are described in Subheading 3. (A). Stability of trypsin derivatives in presence 30% dioxane. (♦) Optimized sulfate-dextran support immobilized derivative at pH 7.0. (■) Optimized sulfate-dextran support immobilized at pH 5.0. (A) Carboxymethyl support immobilized derivative at pH 7.0. (•) Carboxymethyl support immobilized derivative at pH 5.0. (B) Stability of chymotrypsin derivatives in presence 50% dioxane. (♦) Optimized sulfate-dextran support immobilized derivative pH 7.0. (A) Optimized sulfate-dextran support immobilized derivative pH 5.0. (■) Carboxymethyl support immobilized derivative at pH 7.0. (•) Carboxymethyl support immobilized derivative at pH 5.0.

4. Notes

1. PEI viscosity is high, so the 10% PEI solution (w/v) requires a gentle stirring during preparation.

2. Viscosity of sulfate-dextran solution is high. It is therefore necessary to wash exhaustively with abundant water to eliminate excess.

3. To immobilize on PEI supports it is necessary to first wash the PEI support with immobilization buffer in order to balance the pH on the support and prevent the buffer effect caused by the ionic polymer, which could change the pH, and avoid the enzyme immobilization or promote enzyme inactivation.

4. To immobilize on sulfate-dextran supports it is necessary to first wash the sul-fate-dextran support with immobilization buffer in order to balance the pH on the support and prevent the buffer effect caused by the ionic polymer, which could change the pH, and avoid the enzyme immobilization or promote enzyme inactivation.

5. The polymer size is an important parameter because a large size is necessary to wrap up the proteins. Moreover, the polymer size is strongly related with protein size.

References

1. Rosevear, A. (1984) Immobilized biocatalysts: a critical review. J. Chem. Technol. Biotechnol. 34B, 127-150.

2. Royer, G. P. (1980) Immobilized enzymes as catalysts. Catal. Rev. 22, 29-73.

3. Klivanov, A. M. (1983) Immobilized enzymes and cells as practical catalysts. Science. 219, 722-727.

4. Hartmeier, W. (1985) Immobilized biocatalysts: from simple to complex systems. Trends Biotechnol. 3, 149-153.

5. Kennedy, J. F., Melo, E. H. M., and Jumel, K. (1990) Immobilized enzymes and cells. Chem. Eng. Prog. 45, 81-89.

6. Katchalski-Katzir, E. (1993) Immobilized enzymes: learning from past successes and failures. Trends Biotechnol. 11, 471-478.

7. Chibata, I., Tosa, T., and Sato, T. (1986) Biocatalysis: immobilized cells and enzymes. J. Mol. Catal. 37, 1-24.

8. Gupta, M. N. (1991) Thermostabilization of proteins. Biotechnol. Appl. Biochem. 14, 1-11.

9. Mateo, C., Abian, O., Fernandez-Lafuente, R., and Guisan, J. M. (2000) Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support Polyethylenimine supports. Biotechnol. Bioeng. 7, 98-105.

10. Pessela, B. C. C., Fernandez-lafuente, R., Fuentes, M., et al. (2003) Reversible immobilization of a thermophilic P-galactosidase via ionic adsorption on PEI-coated sepabeads. Enzyme Microb. Technol. 32, 369-374.

11. Fuentes, M., Maquiese, J., Pessela, B. C. C., Abian, A., Fernandez-Lafuente, R., Mateo, C., and Guisan, J. M. (2004). New cationic exchanger support for reversible immobilization of proteins. Biotechnol Prog. 20, 284-288.

12. Fuentes M., Pessela B. C. C., Maquiese, J., et al. (2004) Reversible and strong immobilization of proteins by ionic exchange on supports coated with sulfate-dextran. Biotechnol Prog. 20, 1134-1139.

13. Batista-Viera, F., Barbieri, M., Ovsejevi, K., Manta, C., and Carlsson, J. (1991) A new method for reversible immobilization of thiol biomolecules based on solidphase bound thiosulfonate groups. Appl. Biochem. Biotechnol. 31, 175-195.

14. Batista-Viera, F., Brena, B., and Luna, B. (1988) Reversible immobilization of soybean amylase on phenylboronate-agarose. Biotechnol. Bioeng. 31, 711-713.

15. Brena, B., Ovsejevi, K., Luna, B., and Batista-Viera, F. (1993) Thiolation and reversible immobilization of sweet potato amylase on thiosulfonate agarose. J. Mol. Catal. 84, 381-390.

16. Chibata, I. and Tosa, T. (1976) Industrial applications of immobilized enzymes and immobilized microbial cells. In: Applied Biochemistry and Bioengineering: Immobilized Enzyme Principles vol 1 (Wingard, Katchalski, Goldstein, eds.) London, pp. 239-260.

17. Torres, R., Pessela, B. C. C., Mateo, C., et al. (2004) Reversible immobilization of glucoamylase by ionic adsorption on sepabeads coated with polyethyleneimine. Biotechnol. Progr. 20, 1297-1300.

18. Tammi, M., Ballou, L., Taylor, A., and Ballou, C. (1987) Effect of glycosylation on yeast invertase oligomer stability. J. Biol. Chem. 262, 4395-4401.

19. Chu, F. K., Watorek, W, and Maley, F. (1983) Factors affecting the oligomeric structure of yeast external invertase. Arch. Biochem. Biophys. 223, 543-555.

20. Reddy, A. V., MacColl, R., and Maley, F. (1990) Effect of oligosaccharides on oligomeric structures of external, internal and deglycosylated invertase. Biochemistry. 29, 2482-2487.

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